Retinal Binding Site Research Articles

Schiff's base formation in the lens protein γ-crystallin

Uniquely among the soluble lens-specific proteins, γ-crystallin is capable of binding the strongly chromophoric aldehyde retinal. a role for γ-crystallin in protecting lens components from toxic aldehydes resulting from membrane oxidation is proposed and a molecular model of the probable interaction site is presented. The sequence of a tetrapeptide at this site is identical to that of the retinal binding site of bacteriorhodopsin.

Structural studies on membrane-bound bovine rhodopsin.

Our previous work has shown that the treatment of bovine rhodopsin with the proteolytic enzyme papain gives rise to a cleaved, but fully functional, complex consisting of three fragments, H, M and L (heavy, medium and light), held together by strong non-covalent forces. By using some of the chemical and physical differences between the three fragments, a protocol for the preparative isolation of each fragment was devised. Purified M-fragment, which had been radiochemically labelled at the retinal-binding site was treated with CNBr and the mixture subjected to a multi-step separation to furnish a retinyl peptide. The sequence analysis of the latter showed that the retinal-binding lysine residue was located at position 296 from the N-terminal of rhodopsin (or residue 53 from the C-terminal). In order to ascertain the position of the cytoplasmic loop which exists between the M- and L-fragments, radiochemically labelled L-fragment was isolated from the cleaved complex. The purified L-fragment was shown to consist of two populations of peptides which were produced by the action of papain on the bonds between Lys-311 and Gln-312 and between Gln-312 and Phe-313.

The carboxyl-terminal one-third of bovine rhodopsin: Its structure and function

The covalent sequence of the carboxyl-terminal one-third of bovine rhodopsin has been determined. It is 108 amino acids in length and consists of three hydrophilic regions exposed to the aqueous environment at membrane surfaces and two hydrophobic regions which penetrate the disc membrane lipid bilayer. This carboxyl-terminal third of rhodopsin contains the retinal binding site, sites of phosphorylation, the fast-reacting sulfhydryl group and the thermolytic fragment F2. Hydrophobic helices in the sequence have been predicted by a new method developed for application to membrane proteins and a hypothetical “helical hairpin” space-filling working model has been constructed.

Functional domains of rhodopsin

The energetic coupling between the retinal binding site domain and the hydrophilic surface domain of rhodopsin has been studied by measuring the effect of papain proteolysis on the Meta I-Meta II transition kinetics and the ability of rhodopsin to participate in the light activation of the ROS phosphodiesterase. While proteolysis had little effect on the Meta I-Meta II transition kinetics, it had appreciable effects on the level of phosphodiesterase activity. These results support the idea that the hydrophobic retinal binding and hydrophilic surface domains are functionally distinct. It is suggested that light driven conformation changes in the retinal binding site generate the formation of the enzyme interaction site in the hydrophilic domain.

42] Cyanoborohydride reduction of rhodopsin

Publisher Summary This chapter discusses the cyanoborohydride reduction of rhodopsin. The dark-adapted form of rhodopsin resists reduction, apparently because the binding site is deep in a hydrophobic pocket of the molecule, inaccessible to small hydrophilic molecules. This contrasts with the more accessible retinal binding sites of cone pigments, which can be directly reduced by sodium borohydride. Cyanoborohydride is a more hydrophobic borohydride derivative. It serves to penetrate into the hydrophobic core of the rhodopsin molecule and reduce the retinal linkage to lysine. The usefulness of the reaction, beyond confirming that the dark-adapted form of rhodopsin has a retinal Schiff base, is that it chemically binds the retinal to its original binding site in a secondary amino linkage that is not subject to hydrolysis or transaminization to other amino groups in the process of fragmentation and purification. Two additional advantages of cyanoborohydride are, first, that it has a greater specificity for Schiff base reduction relative to aldehyde reduction and, second, that it has a greater stability at neutral and acid pH values.

Synthesis of retinylidene-peptides of bacteriorhodopsin fromHalobacterium Halobium

The purple membrane from Halobacterium Halobium contains a single protein to which retinal is bound via a Schiff-base linkage to a lysine residue. On light excitation, the chromophore undergoes a photocycle during which protons are pumped across the membrane (for a review see (I)). Three observations distinguish the ground state of bacteriorhodopsin from that of protonated retinylidene azomethines: I. the bathochromic shift of the absorption maximum (440 ~570 nm), 2. the stabilization of the 13-cis over the all-trans conformation and 3. the induction of chirality in the chromophore. In order to elucidate these protein-retinal interactions we synthesized retinylidene-peptides with sequences simulating the retinal binding site: (TfaGly-Lys(Tfa)-Lys-Phe-Tyr-Ala (I), TfaGly-Asp-Ala-Lys(Tfa)-Lys-Phe-Tyr-Ala (II) and TfaGly-Val-Ser-Asp-Pro-Asp-AlaLys(Tfa)-Lys-Phe-Tyr-Ala (III). To insure the specific binding of retinal to Lys-41 all other amino functions were protected by the trifluoroacetyl group which is stable under the final HF-cleavage conditions. The spectroscopic properties of the retinylidene peptide III are strikingly different from other azomethines of retinal. This peptide, on reaction with retinal reveals a strong red shift in the absorption spectrum indicating a negatively charged group in the neighbourhood of the azomethine. Additionally an interaction of the bound retinal with tyrosine can be shown. Furthermore, the induction of chirality, induced by these interactions is indicated by the results of circular dichroic measurements. Our experiments demonstrate that the peptides II and III, being only 5% of the amino acid sequence of bacteriorhodopsin display already some features of the intact bacteriorhodopsin molecule.

Specific photoisomerization of retinal in squid rhodopsin and metarhodopsin

The composition of retinal isomers in the photosteady-state mixtures formed from squid rhodopsin and metarhodopsin was determined by high-pressure liquid chromatography. A large amount of 9- cis-retinal was obtained at liquid N 2 temperature when rhodopsin was irradiated with orange light, but only small quantities of 9- cis-retinal were obtained at 15°C. Scarcely any 9- cis-retinal was produced from metarhodopsin by irradiation at liquid N 2 temperature. A large quantity of 7- cis-retinal was found in the photoproduct of rhodopsin irradiated at solid carbon dioxide temperature, but not at 15°C and liquid N 2 temperature. 7- cis-Retinal was not produced from metarhodopsin at any temperatures. These results indicate that the photoisomerization of retinal is regulated by the structure of the retinal-binding site of this protein. The formation of 9- cis- and 7- cis-retinals is forbidden in the metarhodopsin protein.

PURPLE MEMBRANE ANALOGS SYNTHESIZED FROM C17 ALDEHYDE

Abstract— All‐trans, 11‐cis and 9‐cis isomers of the C17 aldehyde analogs of retinal bound with purple membrane apoprotein, probably through a Schiff base linkage at the normal retinal binding site. The complex formed from C17 aldehyde and purple membrane apoprotein was slowly decomposed by 10mM hydroxylamine. The C17 aldehyde competitively inhibited the regeneration of purple membrane from all‐trans‐retinal and purple membrane apoprotein. The differential ability of the different isomers to inhibit the regeneration suggests that purple membrane has a binding site for the side chain of retinal in addition to the Schiff base binding site.

Photochemical studies of 7-cis-rhodopsin at low temperatures. Nature and properties of the bathointermediate.

The photoreaction of 7-cis-rhodopsin derived from 7-cis-retinal and cattle opsin was studied by low-temperature spectrophotometry. Upon irradiation of 7-cis-rhodopsin at liquid nitrogen temperature (-190 degrees C) with blue light, its spectrum shifted to the longer wavelengths, indicating the formation of a bathoproduct. The bathoproduct thus formed was found to be identical with bathorhodopsin formed from rhodopsin in their spectroscopic, photochemical, and thermal properties. Therefore, we believe that the bathoproduct is, in fact, bathorhodopsin. The fact that 7-cis-rhodopsin can be readily converted to rhodopsin and to 9-cis-rhodopsin shows that the identical retinal binding site of opsin is involved in the three isomeric rhodopsins. These results appear to be consistent with the notion that the chromophore of bathorhodopsin is a twisted all-trans isomer, which is readily obtainable from the 7-cis, 9-cis, and 11-cis isomers.

Thyrotropin releasing hormone: Apparent receptor binding in retina

Thyrotropin releasing hormone (TRH) binds with high affinity to sites on sheep retinal membranes. These sites closely resemble sheep pituitary receptors for TRH, as determined by direct comparison of binding in the two tissues. Specific [ 3H]TRH binding in sheep pituitary and retina was measured relative to blanks containing 1 μ m [3-Me-His 2]TRH, an analog more selective and 10-fold more potent than TRH. Incubations were at 0°C; bound radioactivity was separated by filtration. High affinity retinal TRH binding sites resembled pituitary receptors in (1) equilibrium dissociation constant (about 20–40 n m), (2) rate constant for association (about 1–2 μ m −1 min −1), (3) rate constant for dissociation (about 0·07 min −1), (4) effectiveness of 16 TRH analogs, ranging over six orders of magnitude in potency, in competing for binding. Major differences were (1) the retinal binding sites were present in only one quarter the concentration of pituitary receptors (about 5 pmol/g wet weight vs. 20 pmol/g wet weight) and (2) the pharmacological properties of retinal binding sites were partially obscured by the simultaneous presence of low affinity binding sites, absent in pituitary, with which 6 analogs appeared to compete more potently than for high affinity sites. These findings support the possibility that TRH or a closely related substance acts as a peptide neurotransmitter or neuromodulator in the retina.

Ethanolamine attack of the bovine rhodopsin chromophore

Ethanolamine attacks the rhodopsin chromophore, bleaching the visual pigment in the dark. Rhodopsin breakdown is first order under all conditions and the kinetic constants of breakdow are linear in ethanolamine concentration, suggesting that it is a specific attack on the retinal binding site, rather than a non-specific denaturation of the molecule. The rate of breakdown accelerated markedly at basic pH's in the range of the pK's of the protein's basic amino acids, implying that charge repulsion distorts the rhodopsin molecule, making ethanolamine penetration easier.

RHODOPSIN: ITS MOLECULAR SUBSTRUCTURE AND PHOSPHOLIPID INTERACTIONS*

Abstract—Rhodopsin in retinal rod outer segment disc membranes, was proteolyzed by treatment with papain. This treatment left three fragments of apparent mol wt of 26,000, 19,000 and 10,000 in the membrane. The circular dichroism (CD) of solubilized, proteolyzed rhodopsin, in both the UV and visible spectral regions, was essentially identical to that of native rhodopsin. This indicates that the retinal binding site configuration is essentially unchanged by proteolysis and that the proteolyzed form of rhodopsin retained the helical content of native rhodopsin. Far UV CD measurements on the fragments indicate that the secondary structural features of the proteolyzed complex were largely maintained when the complex was dissociated. This finding suggests that the proteolytic fragments represent independently stabilized domains within rhodopsin.Measurements of the dependence of the activation free energy of the unfolding of opsin (as determined by the rate of loss of regenerability of opsin) and the meta I to meta II transition on the level of phospholipid associated with opsin and rhodopsin. respectively, have allowed for a determination of the mode of stabilization of these proteins by phospholipid. This dependence has been shown to have a linear form for opsin and rhodopsin. Hence, it appears that the stabilization of the tertiary structure of both solubilized opsin and rhodopsin is attributable to the sum of their interactions with individual phospholipid molecules, interacting with the protein in a non‐cooperative manner.

Specificity of the retinal binding site of bacteriorhodopsin: chemical and stereochemical requirements for the binding of retinol and retinal.

The complexes formed from bacteriopsin and various retinyl compounds were analyzed by fluorescence and absorption spectroscopy. The binding of retinol occurs in two steps. In the first reaction the molecule is fixed in the retinal binding site of the protein. In this state, energy transfer from aromatic amino acid residues to the retinyl moiety is observed. all-trans-Retinal and the 13-, 11-, and 9-cis-retinols are bound in the chromophoric site. In the second reaction the cyclohexene ring and the side chain of the retinyl moiety are forced into a planar conformation. This reaction is mediated by a base (B1) with a pK of 3.8 and requires the oxygen atom but not the free hydroxyl group of retinol, indicating interaction with a group AH (pK greater than or equal to 10.5). The ring-chain planarization reaction is blocked for the 9-cis isomer of retinol. Binding studies with bacterioopsin and retinal isomers reveal that, as in the case of the corresponding retinols, B1 mediates ring-chain planarization in the case of the all-trans, 13-cis, and 11-cis isomers but not with the 9-cis isomer. Reconstitution of the purple complex from the intermediate 430-460-nm chromophore requires the presence of a second base (B2) with a pK of 4.6. This reaction is exclusive for all-trans- and 13-cis-retinal

Proteolysis and flash photolysis of bacteriorhodopsin in purple membrane fragments.

Pronase treatment of aqueous suspensions of purple membrane fragments from H.halobium leads to the cleavage of bacteriorhodopsin. The protein fragments remaining in the membrane after treatment with relatively small concentrations of enzyme (2% w/w) in normal daylight range in molecular weight from 20,000--21,000 daltons, indicating that cleavage occurs mainly near the extremities of the protein chain. At higher enzyme concentrations the relative amounts of protein fragments having smaller molecular weight increase. Generally, the relative loss of retinal chromophore is larger than that of protein and thus the retinal binding site seems to be located near one of the chain ends that is cleaved off by enzyme. Irradiation with white light during the time of proteolysis (at both low and high enzyme concentrations) results in extensive cleavage, so that under certain conditions no high molecular weight components can be detected in SDS-polyacrylamide gels. It, therefore, appears that parts of the bacteriorhodopsin chain become more exposed to enzyme digestion when the purple membrane is illuminated. Enzyme treated aqueous purple membrane fragment suspensions still show photocycle activity. The main consequence of proteolysis is a pronounced appearance of biphasicity in the decay of M412 and the regeneration of bR570. Simultaneously the yield of O660 is reduced. As with untreated purple membrane, the correlation between the rates of decay of M412 and regeneration of bR570 is greatest when the yield of O660 is lowest.

Photoreceptor protein from the purple membrane of Halobacterium halobium. Molecular weight and retinal binding site.

The apparent molecular weight of the purple membrane protein of Halobacterium halobium was found to be 20 000 by sodium dodecyl sulfate gel electrophoresis and by gel filtration in sodium dodecyl sulfate. However, the molecular weight value determined by gel filtration in 6 M guanidine was 28 000. To resolve this discrepancy, methods insensitive to or independent of the conformation of the protein were used to estimate the molecular weight. Analytical ultracentrifugation of the sodium dodecyl sulfate-protein complex, peptide mapping, and amino acid analysis all gave values of 25 000 +/- 1000, a figure in agreement with a recent x-ray study. Borohydride reduction was used to attach the retinal cofactor covalently to a lysine residue. After digestion with thermolysin, peptide maps were prepared of the protein labeled at lysine residues with [14C] succinic anhydride both before and after reduction. Comparison of the maps showed one radioactive peptide with changed mobility. This peptide was isolated and shown to have the sequence Val-Ser-Asp-Pro-Asp-Lys-Lys with only one of the two lysine residues alkylated. Solid-phase sequencing showed the succinyl group to be at position 6 and hence the retinal group to be at position 7. It was possible that a small amount of retinal was also bound to Lys-6. There was no apparent homology with the corresponding peptide of vertebrate rhodopsin. No evidence of chain heterogeneity was found by radiochemical peptide mapping and sequence analysis of peptides containing lysine residues indicating that all protein chains of purple membrane are very similar or identical.